Clone 1a4

The following primary antibodies were used: Mouse anti-OCT3/4 (Santa Cruz Biotechnology, Clone C-10, #sc-5279, 1:50), Rabbit anti-CDX2 (BioGenex, Clone EP25, #NU777-5UC, 1:100), Mouse anti-CDX2 (BioGenex, Clone CDX2-88, #MU392A-5UC, 1:100), Rabbit anti-EOMES (Abcam, #ab23345, 1:100), Mouse anti-SMA (SIGMA ALDRICH, Clone 1A4, #A2547, 1:200), Mouse anti-TUJ1 (Biolegend, Clone TUJ1, #801201, 1:200), Goat anti-GATA6 (R&D Systems, #AF1700, 1:100), Mouse anti-5mC (Millipore, Clone 33D3, #MABE146, 1:200), Rabbit anti-H3K27me3 (Millipore, #07-449, 1:100) and Rabbit anti-H3K9me3 (Abcam, #ab8898, 1:100). The secondary antibodies used were: Goat anti-Mouse IgG FITC-conjugated (Thermo Fisher Scientific, #62-6511, 1:50), Goat anti-Mouse IgG Alexa Fluor^R 555 (Abcam, #ab150114, 1:1000), Donkey anti-Goat IgG Alexa Fluor^TM 488 (Thermo Fisher Scientific, #A11055, 1:1000), Donkey anti-Mouse IgG Alexa Fluor^TM 555 (Thermo Fisher Scientific, #A31570, 1:1000), Donkey anti-Rabbit IgG Alexa Fluor^TM 555 (Thermo Fisher Scientific, #A31572, 1:1000), and Donkey anti-Rabbit IgG Alexa Fluor^TM 488 (Thermo Fisher Scientific, #A21206, 1:1000).

For protein expression analysis, a conventional SDS–PAGE followed by protein blotting strategy was performed as described previously [29 (link)]. The following antibodies were used: rabbit anti-GAPDH (1:5000, clone GA1R, Thermo Fisher Scientific), rabbit anti-αSMA (1:800, clone 1A4, Biolegend), mouse anti-fibronectin (1:700, polyclonal, Abcam), rabbit anti -TAK (polyclonal 1:1000), rabbit anti-phospho-TAK (polyclonal, 1:1000), anti-Smad2 (clone D43B4, 1:1000) and anti-phosho-Smad2 (clone 138D4, 1:1000, all Cell Signaling Technology, Danvers, MA, USA). Protein abundance was normalized to GAPDH levels. Results were analyzed with ImageJ software (NIH, Bethesda, MD, USA) according to methods for Western Blot densitometry band quantification through image analysis software with background subtraction [38 (link)].

After stimulation, cells were fixed with 4% paraformaldehyde for 5 min at room temperature, permeabilized with 0.01% Triton X-100 for 5 min and blocked with 5% BSA in PBS for 20 min. Specimens were incubated with primary mouse anti-β-catenin (1:500, clone CAT-5H10, Thermo Fisher Scientific, Waltham, MA, USA) and an anti-αSMA antibody (1:200, clone 1A4, Biolegend, San Diego, CA, USA) at room temperature, followed by the secondary AlexaFluor555 goat anti-mouse antibody (1:8000) and phalloidin (1:1000, all Thermo Fisher Scientific) at room temperature for 1 h. Hoechst (1:1000, Thermo Fisher Scientific) was used to label the nuclei. Immunofluorescence was analyzed using Leica DM5500 B fluorescence microscope with DFC365 FX camera (Leica Microsystems, Wetzlar, Germany).